Grafted Photo-Polymerized Monolithic Column

ABSTRACT

The present invention relates to the fabrication of a grafted, UV photo-polymerized silica-based monolithic column and the use of such column for the extraction of DNA. In one embodiment, a method is provided for fabricating a silica-based monolithic column, wherein a vessel is filled with a polymerization mixture that is formed into monolithic solid phase for DNA extraction through in situ photo-polymerization.

The present invention claims priority from U.S. Provisional ApplicationSer. No. 60/656,998, filed Feb. 28, 2005, and from U.S. ProvisionalApplication Ser. No. 60/740,977, filed Nov. 30, 2005, both of which areincorporated herein by reference herein in their entirety.

BACKGROUND OF THE INVENTION

DNA extraction is a sample preparation technique often utilized inclinical and forensic applications to purify and concentrate DNA forgenetic analysis from small volume samples that typically are dilute orbiologically complex, e.g. blood. Significant effort has been investedin the last two decades into devising methods that reduce the amount ofsample required for genetic analysis, often to address the needs ofclinical and forensic communities. One such method has involved adaptingtraditional genetic analysis methodologies to a microscale format. Theminiaturization of sample preparation techniques, including DNAextraction, has been included in the move towards microscale analysis.Such miniaturization has been found to minimize sample handling andcontamination, as well as helping to reduce analysis time. Solid phaseextraction (SPE), the current DNA sample preparation technique of choicein clinical and forensic laboratories, is among the techniques that havebeen miniaturized for microscale analysis. Micro-SPE (μSPE) columns havebeen developed in both capillaries and microdevices such as microfluidicchips (Tian et al., Anal. Biochem. 2000, 283, 175-191; Wolfe et. al.Electrophoresis 2002, 23, 727-733; Breadmore et al. Anal. Chem, 2003,75, 1880-1886).

In the presence of a chaotropic solution, nucleic acids bind avidly to ahydrophilic silica surface. This has been described previously andrepresents the chemical basis for the most common form of SPE for DNAsamples. The most widely used silica-based SPE column for DNA extractionis fabricated using silica-based particles or beads (Melzak et al. J.Colloid Interf. Sci. 1996, 181, 635-644). DNA extraction andpurification has been achieved with good efficiency in microscaleformats utilizing silica beads in μSPE columns. Issues ofreproducibility, however, have resulted from the inability to completelyimmobilize the silica beads within the column (Wolfe et al.Electrophoresis 2002, 23, 727-733). This problem has been addressedusing the dual weir-type approach described in Anal. Chem. 2000, 72,585-590 and a bead immobilization method described in Anal. Chem. 2003,75, 1880-1886. In the latter method, silica beads were packed into themicrochannels of a microfluidic glass chip and immobilized with a“nano-glue” comprised of a tetraethoxyorthosilicate (TEOS) basedsol-gel. This provided a continuous and stable solid phase μSPE columnfor DNA extraction.

Despite improvements made to silica bead based μSPE columns, fabricationof this type of solid phase column within microdevices, particularlythrough bead packing, has several distinct disadvantages. First, theadditional processes involved in filling microchannels on devices suchas microfluidic chips with silica beads increases the fabrication timefor such solid phase columns. Second, when using microfluidic chips forextraction, chip-to-chip extraction reproducibility, while somewhatimproved through bead immobilization, continues to be a significantproblem. Third, while the surface area available for DNA binding isenhanced by decreasing the bead diameter, smaller diameter beads (e.g.,5 μm) are more difficult to contain, resulting in higher back pressureswhich limit μSPE columns in microdevices to relatively low flow ratesand low binding capacity. Several prior art examples have demonstratedthat bead packing problems can be eliminated by providing a high surfacearea-to-volume ratio in a μSPE chamber through the etching of pillars inthe chamber during fabrication (Cady, N. C.; Stelick, S.; Batt, C. A.Biosens. Bioelectron. 2003, 19, 59-66; Christel, L. A.; Petersen, K.;McMillan, W.; Northrup, M. A. J. Biomed. Eng. 1999, 121, 22-27). Whilethis increases the surface area for DNA binding and provides a regulararray for reproducible chromatography, complex fabrication requirementsand cost make these microdevices less attractive. Moreover, a largevolume of elution buffer (greater than 50 μL) is required to elute thebound DNA from the columns of these microdevices, creating potentialdifficulties with downstream processing (e.g., PCR).

The fabrication of silica- and organic polymer-based rigid, porousmonolithic columns has been reported as an alternative to using silicabeads in HPLC and SPE applications and has provided new possibilitiesfor the fabrication of μSPE columns in microdevices. Such monolithiccolumns are fabricated in situ by thermal- or photo-inducedpolymerization of a solution of monomer, initiator, and porogenicsolvent. The resulting solid phase comprises pores in the nanometer tomicron size range with a continuous interconnected network of channels.The advantages of in situ polymerization, including pore size control,high flow-rate and large mass-transfer, have allowed them to besuccessfully used in capillary electrochromatography andpre-concentration applications, e.g., chemical compounds, peptides andproteins.

Thermally-induced polymerized monolithic columns have been demonstratedas a functional medium for DNA separations by HPLC on commercialflat-disk CIM® (BIA Separations) monolith columns. DNA purification andseparation have also been performed on bacterial and yeast genomic DNAin these columns; however, these columns showed low extractionefficiencies and required a high salt and high pH buffer for DNArelease, which has been found to interfere with downstream processing,e.g. PCR. Moreover, thermally-induced polymerization does not ensure theaccurate placement of monolithic columns within the architecture ofmicrodevices. By contrast, UV initiated photo-polymerized monolithiccolumns can be formed within specified spaces. As a result, both silica-and organic polymer-based photo-polymerized monolithic columns have beenincorporated into microdevices (Morishima et al. J. Anal. Chem. 2001,73, 5088-5096).

While silica-based monolithic columns have been found to be functionalfor binding and extracting DNA, the use of certain silica-based monomersas part of the initial polymerization mixture has been problematic.Tetraethylorthosilicate (TEOS)-based monolithic columns have been foundnot to yield extraction efficiencies comparable to columns created withsilica beads, due mainly to the difficulty in controlling pore sizewithin such silica-based sol gel columns which, in turn, inhibits fluidflow. Silica-based monolithic columns reported by Ferrance et al. Anal.Chim. Acta 2003, 500, 223-236, were produced usingtetramethoxyorthosilicate (TMOS) monomers and a porogen to provide theappropriate pore size, but these columns could not easily be localizedwithin microdevices.

None of the methods described above provides the important advantages ofthe fabrication method for a grafted UV photo-polymerized silica-basedmonolithic column. These advantages include increased column capacityand efficiency for DNA extraction resulting in significantly higher DNAyields from very low volume DNA samples of the type encountered inclinical and forensic applications; precise placement of the monolithiccolumn in a capillary or other microdevice, such as a microfluidicmicrochip; minimal reagent volume required to elute DNA from the column;and the ability to use a low ionic strength buffer for the elution ofDNA from the monolithic column, allowing for direct PCR analysis of theextracted DNA without further sample cleaning steps.

SUMMARY OF THE INVENTION

The present invention relates to the fabrication of a grafted, UVphoto-polymerized silica-based monolithic column and the use of suchcolumn for the extraction of nucleic acid. In one embodiment, a methodis provided for fabricating a silica-based monolithic column, wherein avessel is filled with a polymerization mixture that is formed intomonolithic solid phase for nucleic acid extraction through in situphoto-polymerization.

In another embodiment of the invention, a method is provided forfabricating a grafted, UV photo-polymerized silica-based monolithiccolumn, said method comprising the steps of:

(a) providing a vessel;(b) conditioning the interior surface of said vessel by contacting itwith a silica-based flushing solution;(c) preparing a silica-based monomer solution through hydrolysis of asilica monomer;(d) forming a polymerizable mixture by admixing said silica-basedmonomer solution, an initiator material, and a porogenic solvent;(e) introducing said polymerizable mixture into the capillary;(f) initiating the in situ polymerization of said polymerizable mixtureby exposing UV light exposure means to selected portions of the mixture,thereby forming a silica-based monolith column within portions of thecapillary; and(g) flushing a silica-based reagent through said silica-based monolithcolumn, thereby forming the grafted porous silica-based monolith columnwithin portions of the vessel.

In one embodiment of the invention, the vessel is a capillary. In yetanother embodiment of the invention a method is provided for solid phaseextraction of nucleic acid.

The silica-based monolithic column described in the present inventionpossesses enhanced capacity and efficiency for extraction, allowing foran increase in the yield of nucleic acid extracted from very low volumesamples. Additionally, the silica-based monolithic column described inthe present invention allows for nucleic acid to be eluted from thecolumn using a minimal volume of a low ionic strength buffer, whichfurther allows direct PCR analysis of the extracted nucleic acid withoutfurther sample cleaning steps, unlike the elution buffers used withcommercial monolithic affinity columns which typically have a high saltconcentration and/or high pH. Furthermore, the silica-based monolithiccolumn described in the present invention allows for precise placementof the monolithic column in a capillary or other microdevice, such as amicrofluidic microchip.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing the side view of the experimental setup fornucleic acid extraction using a capillary containing a grafted,UV-photopolymerized silica-based monolithic column demonstrating thelocation of the inlet and outlet ports. sol-gel matrix.

FIG. 2A shows a cross sectional view of a capillary device containing agrafted, UV-photopolymerized silica-based monolithic column.

FIG. 2B is a scanning electron micrograph showing a cross sectional viewof the actual internal micro-structure of the grafted,UV-photopolymerized silica-based monolithic column.

FIG. 3 is a graph showing typical DNA extraction profile of a 380 bpfragment of the β-globin gene in human genomic DNA produced using a 10%TMSPM monolith. Legend: 1. TE buffer baseline (from elution step). 2.Loading step baseline. 3. Excess single- and double-stranded DNA(resulting from column overloading). 4. System peak (resulting fromguanidine-isopropanol interaction). 5. Washing step baseline(isopropanol). 6. Mixture of single- and double-stranded DNA in elutionpeak. 7. Scanning electron micrograph of the 10% TMSPM monolith internalmicro-structure.

FIG. 4 is a graph showing the effect of sample loading time on DNAelution profiles, using a 10% TMSPM monolithic column. Columnconditions: length: 12 cm; back pressure: 7 psi; running pressure: 11psi. The inserted graph shows elution peak area versus sample loadingtime, displaying a stable elution peak area after reaching the columncapacity.

FIG. 5 is a schematic of TMOS derivatization on the TMSPM monolithsurface.

FIG. 6 are graphs showing the effect of TMOS A) concentration and B)derivatization time on extraction capacity (n≧3). Column conditions: 10%TMSPM derivatized with: A) various concentration of TMOS for 30 min andB) 85% TMOS for various lengths of time; length: 12 cm; runningpressure: 12 psi.

FIG. 7 are graphs showing the effect of TMSPM concentration onextraction capacity (n=3), with elution profiles produced using A) LIFand B) UV detection. Column conditions: derivatization with 85% TMOS for45 min; length: 12 cm. 23 Wen et al.—DNA Extraction Using a NovelTetramethylorthosilicate-Grafted Photo-Polymerized Monolithic SolidPhase

FIG. 8 is a graph showing the effect of TMSPM hydrolysis time onrelative column capacity (n=3). Column conditions: 17% TMSPM derivatizedwith 85% TMOS for 45 min; length: 12 cm.

FIG. 9 is a graph showing the effect of column length on relative columncapacity (n=3). Column conditions: 17% TMSPM derivatized with 85% TMOSfor 45 min.

FIG. 10 is a graph showing the extraction of pre-purified human genomicDNA using a 2 cm TMSPM/TMOS monolith. Column conditions: 17% TMSPMderivatized with 85% TMOS for 45 min.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below:

As used herein, the term “purify” and like terms relate to an enrichmentof a molecule or compound relative to other components normallyassociated with the molecule or compound in a native environment. Theterm “purify” does not necessarily indicate that complete purity of theparticular molecule will be achieved during the process. A “highlypurified” compound as used herein refers to a compound that is greaterthan 90% pure.

As used herein a “chaotropic agent” is an agent that is capable ofdisrupting the hydrogen bonding between water and nucleic acid andincludes but is not limited to urea, guanidine hydrochloride, andpotassium iodine.

As used herein, the term “derivatization” and like terms refer to theprocess of bonding the molecules of a suitable silica-based monomerreagent to the surface of a silica-based monolithic column. The term“grafted” refers to the state of the silica-based monolithic columnafter derivatization.

As used herein, the term “immobilization” and like terms refer to theattachment or entrapment, either chemically or otherwise, of material toanother entity (e.g., a solid support) in a manner that restricts themovement of the material.

As used herein a “microcolumn” is a matrix comprising pores of apreferred size of about 0.1-15 μm in diameter, more preferably about1-10 μm, most preferably about 4-6 μm. The microcolumn preferably has anaverage cross sectional dimension of about 1 mm² to about 100 μm², morepreferably about 0.5 mm² to about 7,000 μm², and most preferably 0.275mm² to about 30,000 μm².

As used herein a “microchannel” is a passageway (in any form, includinga closed channel, a capillary, a trench, groove or the like) formed onor in a microfluidic substrate (a chip, bed, wafer, laminate, or thelike) having at least one region with a cross sectional dimension ofabout 5 mm² to about 100 μm², preferably about 0.5 mm² to about 7,000μm², and more preferably 0.275 mm² to about 30,000 μm².

A “microfluidic device” is an apparatus or component of an apparatusthat includes at least one microchannel.

As used herein, the term “sol-gel” refers to preparations composed ofporous metal oxide glass structures. The phrase “monolithic column” andlike phrases refer to a solid bonded network of silica prepared by ahydrolysis—condensation polymerization reaction of suitable monomers.The materials used to produce the monolithic column can include, but arenot limited to, aluminates, aluminosilicates, titanates, ormosils(organically modified silanes), and other metal oxides. As used herein a“vessel” is a passageway in any form (including a closed channel, acapillary, pipette, tube, a trench, groove or the like) having at leastone region with a cross sectional dimension selected from a range ofabout 20-500 μm, preferably 50-300 μm, and more preferably about 100-250μm.

The present invention is directed to a method of fabricating a grafted,UV photo-polymerized silica-based monolithic column and the use of suchcolumn for the extraction of DNA and other biologically-activemolecules. The method of the present invention allows for preciseplacement of the monolithic column in a vessel, such as a capillary,pipette, tube, or microchannel on a microfluidic device (e.g., amicrofluidic chip), unlike columns fabricated in such devices throughsilica bead packing. Furthermore, the method described in the presentinvention allows for an enhancement of the ability to extract and purifyDNA from very low volume samples, in particular, complex or crudebiological samples like blood, using SPE. The method described in thepresent invention also allows for direct PCR analysis of DNA extractedfrom the column by eliminating the cleanup steps between extraction andfurther downstream processing through the use of minimal volumes ofelution reagent.

In a preferred embodiment, the monolithic column is used to purifynucleic acid in a micro-total analysis system (μ-TAS). There are manyformats, materials, and size scales for constructing μ-TAS. Common μ-TASdevices are disclosed in U.S. Pat. No. 6,692,700 to Handique et al.;U.S. Pat. No. 6,919,046 to O'Connor et al.; U.S. Pat. No. 6,551,841 toWilding et al.; U.S. Pat. No. 6,630,353 to Parce et al.; U.S. Pat. No.6,620,625 to Wolk et al.; and U.S. Pat. No. 6,517,234 to Kopf-Sill etal.; the disclosures of which are incorporated herein by reference.Typically, a μ-TAS device is made up of two or more substrates that arebonded together. Microscale components for processing fluids aredisposed on a surface of one or more of the substrates. These microscalecomponents include, but are not limited to, reaction chambers,electrophoresis modules, microchannels, fluid reservoirs, detectors,valves, or mixers. When the substrates are bonded together, themicroscale components are enclosed and sandwiched between thesubstrates. A major advantage of using a μ-TAS resides in the fact thatthe monolithic column can be used a part of an integrated system foranalysis.

In accordance with one embodiment, the method of the present inventioncomprises a polymerized silica-based monolithic column. The use ofsilica-based monolithic columns for DNA extraction under chaotropicconditions has been demonstrated by both Wolfe et al. and Ferrance etal. The preparation of such columns has been previously described byothers. A polymerized silica-based monolithic column is typicallyprepared by the room-temperature mixture and polymerization of suitablemonomers (usually metal alkoxides), initiators, and porogens to form aporous, glassy material. In one embodiment, the silica-based monolithiccolumn is prepared using a polymerization mixture comprised of asilica-based monomer solution, a photoinitiator, and a porogenicsolvent. The column is typically prepared by introducing thepolymerization mixture into a vessel such as a capillary, pipette, tube,or microchannel on a microfluidic device.

From the foregoing, it will be seen that one aspect of the presentinvention is directed to the fabrication of a polymerized silica-basedmonolithic column in a capillary. More particularly, the capillaryranges in length from about 2 cm to 12 cm, has an inside diameter of 250μm, and an outside diameter of 365 μm, with one end of the capillaryserving as an inlet port and the opposing end serving as an outlet port.The polymerized silica-based monolithic column is contained within thecapillary and spans a cross sectional dimension of the capillary so thatfluid traversing from the inlet port to the outlet port must passthrough the column. The column present in the capillary may extend theentire length of the capillary or may extend only a partial length ofthe capillary if localized within portions of the capillary during thepolymerization process. Typically, the polymerization mixture isintroduced into the capillary, and then polymerized so that thedimensions of the monolithic column match the original interior space ofthe capillary.

Still another embodiment of the present invention is directed towardsmodification of the surface of a polymerized silica-based monolithiccolumn in a capillary. Breadmore et at. Anal. Chem. 2003, 75, 1880-1886,showed that silica beads modified with tetraethylorthosilicate (TEOS)provided a phase with excellent properties for DNA extraction.Modification of the surface of a polymerized silica-based monolithiccolumn in a capillary typically involves pressure flushing asilica-based reagent through the capillary at room temperature for anindicated period of time. Molecules of the reagent condense on thesurface of the monolithic column increasing the number of silica bindingsites for nucleic acids.

As shown in FIG. 1, one embodiment of the grafted, UV photo-polymerizedsilica-based monolithic column 1 of the present invention comprises acapillary 2. An inlet port 3 and outlet port 4 are in fluidcommunication with the capillary 2. A cross sectional view of thecapillary 2 is depicted in FIG. 2. FIG. 2 demonstrates that thecapillary 2 is filled with the silica matrix comprising the monolithiccolumn 1. The silica matrix spans a cross sectional dimension of thecapillary, so that a fluid traversing from the inlet port 3 to theoutlet port 4 must pass through the monolithic column 1. FIG. 2A shows ascanning electron micrograph of the actual monolithic column 1 internalmicro-structure.

To prepare the grafted, UV photo-polymerized silica-based monolithiccolumn of the present invention, a silica-based monomer solution isprepared by hydrolyzing a suitable silica-based monomer in an acidicaqueous solution (for example 0.1 M HCl) in accordance with proceduresknown to those skilled in the art. In one embodiment, the silica-basedmonomer solution is prepared through the hydrolysis of3-(Trimethoxysilyl)propyl methacrylate (TMSPM) in an acidic aqueoussolution (for example 0.1 HCl) for five minutes to form the silica-basedmonomer solution. TMSPM is capable of producing a sol-gel monolithiccolumn, but relies upon its acrylate functional groups for formation ofthe final monolithic column by forming a polyacrylate organic matrixaround a silica backbone upon polymerization. TMSPM simultaneouslyprovides the ability to localize the monolithic column within amicrodevice, unlike sol-gel monolithic columns fabricated usingtetramethoxyorthosilicate (TMOS) monomers or through bead packing. Thesilica based monomers appropriate for the present invention include anyalkoxy silane compounds of type R_(n)Si(OR′)_(4n) as described by C. J.Brinker et al. in Sol-Gel Science, Academic Press, Inc., New York, N.Y.,1990, which is incorporated herein by reference. The most commonly usedof these compounds are tetraethylorthosilicate (TEOS),tetramethoxyorthosilicate (TMOS), and poly(ethoxydisiloxane) (PEDS).Other copolymers may also be used including, but not limited to,3-(trimethoxysilyl)propyl methacrylate (TMSPM),3-[tris(trimethylsiloxy)silyl] propyl methacrylate,3-(diethoxysilyl)propyl methacrylate, 3-(dimethylchlorosilyl)propylmethacrylate, 3-(trichlorosilyl)propyl methacrylate, and combinationsthereof.

A polymerization mixture is then formed by admixing the silica-basedmonomer solution with a photoinitiator and a porogenic solvent, inaccordance with procedures known to those skilled in the art. In oneembodiment, the silica-based monomer solution is comprised of TMSPM andis present in the polymerization mixture in a concentration of 17%. Thephotoinitiator, can be, but not limited to, benzophenone,dimethoxyacetophenone, xanthone, thioxanthone, and mixtures thereof witha commercially available mixture.

The porogenic solvent functions to foster the development of a suitablepore structure in the monolithic column during the polymerizationprocess. The porogenic solvent can be, but is not limited to, water,organic solvents, and mixtures thereof. Organic porogenic solvents canbe, but not limited to, hydrocarbons, alcohols, ketones, aldehydes,organic acid esters, soluble polymer solutions, and mixtures thereof. Ina preferred embodiment, the solution is comprised of toluene, as thepreferred porogenic solvent, in a concentration of about 90%.

The polymerization mixture is then introduced into the capillary. In oneembodiment, the capillary is conditioned prior to addition of thepolymerization mixture. Conditioning of the capillary comprises thesteps of rinsing the interior surface of the capillary with a basicsolution (for example, 1 M NaOH), water, an acidic solution (forexample, 0.1 M HCl), and an alcohol solution; flushing the interiorsurface of the capillary with a silica-based flushing solution; andallowing the capillary to dry. In a preferred embodiment, thesilica-based flushing solution is comprised of TMSPM in a concentrationof about 30%.

In situ polymerization of the polymerization mixture within thecapillary is then initiated to form a polymerized monolithic columnwithin the capillary. In one embodiment, polymerization is initiated byexposing the polymerization mixture within the capillary to a UV lightsource, such as, for example, a Model RSM100W 100 W broad UV wavelengthlamp. In a further embodiment, the length of the monolithic columnformed in the capillary through polymerization is controlled byselective exposure of sections of the polymerization mixture within thecapillary to UV light. To achieve selective exposure, a portion of thepolyimide coating on the capillary is removed in the area of the desiredmonolith and other portions of the capillary are covered with a blackcloth during UV exposure to prevent polymerization at these particularlocations in the capillary.

Once the photo-polymerized silica-based monolithic column has beenformed in the capillary, the silica monomer may be grafted onto thesurface of the monolithic column by flushing the column with asilica-based monomer reagent. In a preferred embodiment, thesilica-based monomer reagent is comprised of tetramethylorthosilcate(TMOS), which has enhanced hydrolysis properties under acid-catalyzedconditions and bonds to the surface of the monolithic column at a fasterrate, thereby reducing the total fabrication time for the monolithiccolumn. In a further embodiment, TMOS is preferably present in thesilica-based monomer reagent in a concentration of about 85%. Thereagent is vortexed at room temperature for one minute and then pressureflushed through the column at room temperature. In a further embodiment,the reagent is flushed through the column for 45 minutes. The hydrolyzedTMOS molecules are the bound to the column. In yet another embodiment,TMOS is bound to the column through condensation with unreactedsiloxane/silanol groups that are present on the surface of aphoto-polymerized monolithic column prepared using TMSPM, allowing theformation of a continuous network of silicon dioxide and increasing thenumber of silica binding sites on the surface of the column as a result.

The resulting grafted, UV photo-polymerized silica-based monolithiccolumn possesses enhanced capacity and efficiency for extraction ofnucleic acids. In accordance with one embodiment, a method of extractingnucleic acids from a biological sample comprises a first step ofcontacting the biological sample with a chaotropic agent which lysescellular membranes and releases the cellular nucleic acid sequences. Inone embodiment, the chaotropic agent comprises 6M guanidinehydrochloride prepared in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).After mixing the biological sample with the chaotropic agent, the sampleis directly loaded onto the column of the present invention, theseconditions being conducive for nucleic acid binding to the column. Inaccordance with one embodiment, the length of the column of the presentinvention is decreased from 12 cm to 2 cm. By reducing the length of themonolithic column, the back pressure is reduced allowing higher flowrates through the column. The column is then washed with a suitablesolvent, such as an aqueous/organic (alcohol, acetonitrile) mixture(40-80% organic), to remove unbounded material and the bound nucleicacid sequences are then released from the column by washing the columnwith an appropriate buffer known to those skilled in the art, such asphosphate buffer, citrate buffer, Tris buffer or Chaps buffer, with anionic concentration of less than 150 mM. In one a preferred embodiment,the nucleic acid is released from the column by washing with a bufferthat is compatible with PCR reactions, such as TE buffer.

Binding of nucleic acids to this matrix utilizes the same mechanism asall silica based phases for nucleic acid extraction, with the presenceof the chaotropic salt causing the nucleic acids to adsorb on the silicasurface over a broad chaotropic concentration, pH range, and temperaturewill vary with the phase utilized.

Release from the surface occurs into a low ionic strength aqueous bufferfollowing removal of the chaotropic agent.

Preferably, the temperature at which the binding and release steps areperformed is no greater than about 95° C., more preferably no greaterthan about 70° C., 65° C., 60° C., 55° C., 50° C., 45° C., or 40° C.Most preferably, the same temperatures apply to the entire process forboth the binding and release steps. The release step, or the entireprocess, may even be performed at lower temperatures, such as 35° C.,30° C., or 25° C. Most preferably, the entire process occurs at roomtemperature.

Furthermore, the elution step preferably occurs under conditions of lowionic strength, suitably less than about 500 mM, preferably less thanabout 400 mM, 300 mM, 200 mM, 100 mM, 75 mM, 50 mM, 40 mM, 30 mM, 25 mM,20 mM, or 15 mM, most preferable less than about 10 mM. The ionicstrength may be at least about 5 mM, more preferably at least about 10mM. These ionic strengths are also preferred for the binding step.

The use of such mild conditions for the elution of nucleic acid isespecially useful for extracting small quantities of nucleic acid, asthe extracted DNA or RNA can be transferred directly to a reaction orstorage tube without further treatment steps. Therefore loss of nucleicacid through changing the container, imperfect recovery during furthertreatments, degradation, denaturation, or dilution of small amounts ofnucleic acid can be avoided. This is particularly advantageous when anucleic acid of interest is present in a sample (or is expected to bepresent) at a low copy number, such as in certain detection and/oramplification methods.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following example isgiven to illustrate the present invention. It should be understood thatthe invention is not to be limited to the specific conditions or detailsdescribed in this example.

EXAMPLE Preparation of Grafted, UV-Photopolymerized Silica-BasedMonolithic Column for use in Solid Phase Extraction

Materials and Reagents. Fused-silica capillary (250 μm i.d.×365 μm o.d.)was purchased from Supelco, Inc. (Bellefonte, Pa.).3-(Trimethoxysilyl)propyl methacrylate (TMSPM, minimum 98%) andtetramethylorthosilicate (TMOS, 98%) were obtained from Sigma-Aldrich(Milwaukee, Wis.). Toluene (99.9%), 2-propanol (HPLC grade), guanidinehydrochloride (GuHCl, electrophoresis grade), ethanol (95%) andTris(hydroxymethyl)aminomethane (Tris) were purchased from FisherScientific (Fairlawn, N.J.). EDTA was obtained from American ResearchProducts (Solon, Ohio). Photoinitiator Irgacure 1800 was generouslydonated by Ciba (Tarrytown, N.Y.). All solutions were prepared withnanopure water (Barnstead/Thermolyne, Dubuque, Iowa).

Labeled DNA Fragment Generation. A 380-bp β-globin DNA fragment wasamplified using the polymerase chain reaction (PCR) in a Bio-RadMycycle™ Thermal cycler (Hercules, Calif.) using forward primer taggedwith the fluorescence dye 5-FAM (λ_(ex) 488 nm, λ_(em) 520 nm) (MWGBiotech, High Point, N.C.). Thermocycling conditions were as follows:94° C. for 30 sec, 60° C. for 30 sec and 72° C. for 30 sec (30 cycles)with a 3 min preincubation at 95° C. and a final extension of 1 min at72° C. The purity of the PCR product was assessed by capillaryelectrophoresis (Beckman P/ACE MDQ, Fullerton, Calif.) using LIFdetection. The fluorescently-tagged PCR product was used in preliminarysolid phase extraction experiments to allow sensitive on-line detectionof the profiles achieved using a capillary electrophoresis instrumentequipped with LIF detection.

Preparation of Photo-Polymerized Monolith. The internal wall surface ofa 250 μm i.d. fused silica capillary was first treated with3-(trimethoxysilyl)propyl methacrylate (TMSPM). Briefly, the capillarywas rinsed with 1 M NaOH for 15 min, water for 15 min, 0.1 M HCl for 15min, and finally 95% ethanol for 5 min at 0.1 mL/min. A 30% (v/v) TMSPMsolution (in 95% ethanol), adjusted to pH 4 with acetic acid, wasflushed through the capillary at 3 μL/min for 60 min using a syringepump (KD Scientific, Holliston, Mass.) and subsequently dried under astream of nitrogen overnight. The treated capillary was left at roomtemperature for at least one day before use.

The monomer solution was prepared as previously reported by Dulay etal., Anal. Chem, 2003, 73, 3921-3926. A monomer solution consisting of85% (v/v) TMSPM and 15% (v/v) 0.1 M HCl was stirred at room temperaturein the dark for 20 min. The sol-gel solution was prepared with 10% (v/v)monomer solution, 90% (v/v) toluene and 5% (w/v) photo-initiatorIrgacure 1800 and stirred in the dark at room temperature for 5 min. Thetreated capillary was then filled with the sol-gel solution and exposedto UV light using a 100 W broad UV wavelength lamp (Model RSM100W,Regent Lighting Corp. Burlington, N.C.) for 5 min to initiatepolymerization. The monolith length was controlled by removing a portionof the capillary polyimide coating. The detection window fabricated onthe capillary by removal of the polyimide coating was covered with ablack cloth during UV exposure to prevent polymerization at thisparticular location. After polymerization, the capillary was installedinto the capillary electrophoresis instrument cartridge and ethanol wasflushed through the column to visualize the monolith and remove excessmonomer reagent. The minimum pressure required to flow solution throughthe monolith is referred to as the back pressure through the column inthe following sections.

TMOS Derivatization of Monolith. Once the monolith was formed, thesurface was modified by treatment with various concentrations of TMOS.For example, an 85% TMOS solution was prepared with 85% (v/v) TMOS and15% (v/v) 0.1 M HCl. The TMOS:HCl ratio was varied accordingly fordifferent TMOS concentrations. The solution was vortexed at roomtemperature for 1 min. The monomer solution was then pressure-flushedthrough the monolithic capillary (12 psi) at room temperature for theindicated period of time.

Solid Phase Extraction Procedure. PCR-amplified 380-bp human genomicβ-globin fragment products were diluted 5-fold in 7.5 M guanidinehydrochloride prepared in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).Human genomic DNA, purified from human blood (60 μg/mL, A260/A280ratio=1.795), was diluted 20-fold in 6 M guanidine hydrochloride buffer(pH 6). It is noteworthy that the samples were diluted in guanidinehydrochloride solutions of different starting concentrations due to thedifference in dilution factors, to achieve a final guanidineconcentration of 5.7M.

Before extraction, the SPE column was equilibrated with 6 M guanidinehydrochloride prepared in TE buffer for 10 min. Both sample andequilibration solutions were adjusted to pH 6 using 0.1 M HCl. DNAsamples were loaded onto the column using pressure injections forvarious injection times. A wash step was subsequently performed using80% (v/v) 2-propanol in water to remove unbound DNA andproteins/contaminants then the DNA was eluted with TE buffer. Blankexperiments were performed for each column formulation investigated, byreplacing the DNA sample volume with water. All extraction experimentswere performed at room temperature.

To compare the DNA binding data obtained from the various monolithformulations investigated, the columns were systematically overloadeduntil reaching constant DNA elution peak areas. Excess DNA (not bound tothe monolith), in the load and washing steps, and extracted DNA, in theelution step, were detected online and used to calculate the relativecolumn capacity and extraction efficiency. For the purpose of thispaper, the relative column capacity is defined as the maximum elutionpeak area obtained after overloading the monolithic column. The relativeextraction efficiency is defined as the ratio of the elution peak areato the sum of load, wash and elution peak areas when the column was notoverloaded.

DNA extraction of pre-purified human genomic DNA and whole bloodsamples. Extraction of pre-purified DNA and whole blood were performedusing the TMSPM/TMOS monolith and the QIAamp® DNA mini kit (QIAGEN,Valencia, Calif.). In order to compare the efficiency of the twoextraction methods, the same amount of DNA was loaded onto the columns.To do so, a syringe pump (KD Scientific, Holliston, Mass.) was used todeliver the DNA load solution at a constant flow rate (3 μL/min).Pre-purified human genomic DNA was obtained from blood (60 μg/mL,A260/A280 ratio=1.795) and 120 ng DNA were loaded onto the monolith andQIAGEN columns. Whole blood sample was chemically-treated prior toextraction to release DNA from the white blood cells. For theextractions performed on the monolith, 2.5 μL whole blood was incubatedwith 585 μL load buffer (6 M Gu-HCl, 10 mM Tris-HCl, 1 mM EDTA, pH 5.8)and 15 μL of a 10 μg/μL proteinase K solution in a water bath at 56° C.for 10 min. 20 μL of the digested blood sample were loaded onto themonolith, corresponding to 83 nL whole blood and 4.9 ng DNA. For theextractions performed using the QIAGEN kit, whole blood was preparedaccording to the manufacturer's guidelines. Due to the low volume ofwhole blood loaded onto the QIAGEN column (83 nL), the blood sample wasdiluted in PBS buffer (2.5 μL blood in 600 μL PBS) prior to extraction.DNA was subsequently extracted from the monolith by washing awayproteins and contaminants using 30 μL of a 80% (v/v) 2-propanol inwater, and eluting DNA using 40 μL of TE buffer at a 3 μL/min flow rate.DNA extraction from the QIAGEN column was performed as suggested by themanufacturer. For both extractions, the amount of DNA present in theelution fractions was subsequently quantified.

DNA Quantification. Quantification was performed only for the extractionof purified human genomic DNA samples. Load, wash and elution fractionswere collected, and the amount of DNA in each fraction was subsequentlyquantified using the PicoGreen® assay according to the manufacturer'sguidelines.

Results and Discussion

DNA Extraction Using a TMSPM-based Monolith. In the presence of achaotropic solution, nucleic acids bind avidly to a hydrophilic silicasurface. This has been described previously (Tian, H.; Hühmer, A. F. R.;Landers, J. P. Anal. Biochem. 2000, 283, 175-191), and represents thechemical basis for the most common form of DNA purification viainteraction with a solid phase surface (Boom, R. et. al. J. J. Clin.Microbiol 1990, 28, 496-503). While silica beads represent the mostcommon form of silica used for this purpose, it is feasible to utilizeother forms. This was shown by the reported use of silica sol-gelstructures for DNA capture under chaotropic conditions by both Wolfe etal. Electrophoresis 2002, 23, 727-733), and Ferrance et al. (Anal. Chim.Acta 2003, 500, 223-236). The silica sol-gel monoliths were found to befunctional for binding and extracting DNA—however, Wolfe et al. foundthat the tetraethyl orthosilicate (TEOS)-based sol-gels did not yieldextraction efficiencies comparable to silica beads, mainly because thesol-gel pore size was difficult to control, thereby inhibiting fluidflow through the matrix13. The sol-gel reported by Ferrance et al. wasproduced using tetamethoxyorthosilicate (TMOS) monomers and a porogen toprovide the appropriate pore size, but these could not easily belocalized within the microdevices. 3-(Trimethoxysilyl)propylmethacrylate (TMSPM) is a silica monomer containing an acrylate group,which can also produce a sol-gel monolithic structure, but it relies onthe acrylate functional groups for formation of the final monolith. Uponpolymerization, this monomer forms a polyacrylate organic matrix arounda silica backbone, while providing the ability to localize the monoliththrough UV initiated polymerization. The stability of this monolith, theability of the silica surface to bind DNA, and the tunable flowcharacteristics of these gels were first investigated to determinewhether they could function as a suitable stationary phase for DNAextraction in microdevices.

Initial investigations were performed using a 12 cm long monolithiccolumn fabricated in a fused silica capillary. A fluorescently-labeledPCR-amplified DNA fragment was used in these preliminary experiments.While we understand that a single fragment of low molecular weightcannot be viewed as representative of the heterogeneous character ofhuman genomic DNA, it functions as a simple model to assess thepotential of the monolith for DNA extraction. In addition, we utilizedcapillary electrophoresis (CE) instrumentation equipped with LIFdetection as the platform to evaluate the chromatography (DNA load, washand elution steps) involved in the DNA extraction. The reasons for thisare 1) it provides an excellent platform for control of small-volumesamples and reagents; and 2) it has a sensitive, built-in LIF detectionsystem. A typical extraction profile obtained from the CE using the 380bp fragment of the β-globin gene amplified from human genomic DNA as thesample is presented in FIG. 3. The baseline shifts observed in thisprofile (numbered 1, 2 and 5) are due to the different buffers used inload, wash and elution steps having different optical properties. Thesystem peak (number 4), also present in blank profiles (data not shown),is assumed to result from the interaction between guanidine andisopropanol occurring when load and wash buffers come in contact. Theextraction of a PCR primer sample showed that single-stranded DNA(ssDNA) could also be extracted using the TMSPM monolith (data notshown). Both double-stranded DNA (dsDNA) and ssDNA were thereforeassumed to be present in the excess DNA eluted during the wash step(peak number 3) and in the DNA detected during the elution step (peaknumber 6). In the following experiments, only the elution portion of theextraction profile will be displayed as the efficiency of the monolithswill mainly be evaluated by their relative column capacity (obtainedfrom the elution peak area).

The effect of the TMSPM monomer concentration on the stability and backpressure was examined over a concentration range of 7% to 21%. Low TMSPMconcentrations (<10%) led to the formation of monoliths with poorstructural stability. Increasing the monomer concentration led to theformation of small pores and resulting in a back pressure that increasedalmost linearly with increasing monomer concentration (5 psi for 7%TMSPM; 16 psi for 17% TMSPM). At TMSPM concentrations greater than 19%,the back pressure was too high to effectively pump solution through themonolithic column using the capillary electrophoresis instrumentinternal pressure module (in accordance with previously reporteddata42). Although the extraction efficiency improved with increasingmonomer concentration as a direct result of the smaller pores and highersurface area available for DNA binding (data not shown), the increasedback pressure ultimately led to slower flow rate and longer analysistime. A monomer concentration of 10% (back pressure of 7 psi) allowedfor relatively high flow rates (up to 700 μL/hour) and was selected toevaluate the extraction efficiency of the monolithic column.

Sample containing different masses of DNA (380 bp β-globin fragment)were loaded onto the column using a constant pressure (11 psi) butvarying the injection time (FIG. 4). The longer loading time andsubsequent increase in the mass of DNA loaded onto the column, resultedin an increase in the elution peak area, which reached a maximum atapproximately 2 min (indicated on the inserted graph by the plateauformed from 2 min onwards). The large amount of unbound DNA that exitsthe column during the wash step and the relatively small elution peaksuggested that DNA had a weak affinity for the TMSPM-based monolith. Onepossible explanation for this observation is that a substantial portionof silica binding sites are buried within the highly cross-linkedorganic polymer matrix and inaccessible to the DNA. It was clear thatthe column capacity had to be enhanced by modifying the monolithsupport.

Modification of the TMSPM Monolith with TMOS. Breadmore et al. (Anal.Chem, 2003, 75, 1880-1886) showed that silica beads derivatized withtetraethyl orthosilicate (TEOS) provided a phase with excellentproperties for DNA extraction. They utilized the TEOS to hold the silicabeads in place, but did not elaborate on whether the silica surface ofthe beads, the TEOS derivatization, or both, provided the DNA extractionsites. For this work, it was hypothesized that the characteristics ofthe TMSPM monolith could be improved by derivatizing it with asilica-based reagent. While both tetramethyl orthosilicate (TMOS) andTEOS were candidates for the derivatization of the TMSPM monolith, TMOSwas selected due to its enhanced hydrolysis properties underacid-catalyzed conditions43. The size of the alkoxy groups present onthe organosilane is, indeed, known to affect the hydrolysis rate, whichis 10-times slower for TEOS than TMOS.

Upon acid hydrolysis, the hydrolyzed TMOS (Si(OCH₃)₄) molecules willcondense with the unreacted siloxane/silanol groups present on themonolith surface, as displayed in FIG. 5. The reacting TMOS should forma continuous network of silicon dioxide which would increase the numberof silica binding sites on the monolith surface. The TMOS hydrolysisrate is dependent on the water content in the monomer solution, and highH₂O:Si ratios are expected to favor TMOS hydrolysis. However, high watercontent will also inhibit siloxane bond formation during thecondensation reaction, which will favor self-condensation of TMOS overTMOS-monolith condensation. Consequently, decreasing the water contentis key to reducing TMOS hydrolysis and self-condensation rates, therebyincreasing the reaction with the monolith. The effect of water contenton the derivatization efficiency was investigated using H₂O:Si ratios inthe monomer solution of: 1, 1.5, 2, 4 and 8, corresponding to TMOSconcentrations of 89%, 85%, 80%, 68% and 52%, respectively; the acidcatalyst concentration was varied to maintain a pH of 1.0. Thederivatization time was initially set at 30 min to achieve comprehensivecoverage of the monolith surface with TMOS. Results showed that the useof the TMOS sol-gel derivatization significantly increased the relativecapacity of the monolith column for DNA extraction, particularly whenconcentrations in the 80%-89% range were employed (FIG. 6A). However,the higher the TMOS concentration the higher the pressure generated inthe capillary. Microscopic examination of the monolith suggested thatthis may be due to the reaction proceeding rapidly at the highconcentrations, since only part of the monolith appeared to bederivatized with TMOS (visually identifiable by a change in color).Depending on the TMOS concentration used, the color change was typicallyobserved in the first 2-7 cm of the monolith. The higher concentrationsgave a shorter derivatized region, but one that presumably had greatersurface coverage and smaller pores, which led to an increased backpressure. Only about 2 cm of monolith was derivatized when 89% TMOS wasused, which may explain the corresponding lower relative column capacitycompared to that obtained using 85% TMOS, which covered 2.5-fold more ofthe monolith (˜5 cm). From these observations and the data given in FIG.6A, the largest column capacity was obtained using 85% TMOS, which wastherefore selected for further optimization experiments.

The time needed for effective TMOS derivatization of the sol-gel surfacewas also investigated over the time range of 0 to 60 min (FIG. 6B).Although even short derivatization ties (15-30 min) were found tosignificantly improve the extraction efficiency compared to anunderivatized column, the best capacity and extraction efficiency wereproduced with a 45 min derivatization time. It is noteworthy thatderivatization times greater than 60 min led to capillaries that werecompletely occluded. TMOS derivatization was found to enhance therelative column capacity by about 60-fold (elution peak area=3,000±3,000using the underivatized monolith versus 1,834,000±70,000 using theTMSPM/TMOS column) and the relative extraction efficiency byapproximately 40% (48.5%±1.8% with the underivatized monolith versus94.0%±1.3% with the TMSPM/TMOS column). The extraction reproducibilitywas also acceptable, with run-to-run relative standard deviation (RSD)using a single column below 12% and capillary-to-capillary RSD below11%. As a result of these optimization experiments, the optimal relativecolumn capacity and extraction efficiency was obtained using a monolithderivatized with 85% TMOS for 45 min at room temperature.

Effect of TMSPM Monomer Concentration on DNA Extraction. The monolithutilized in the derivatization optimization experiments was producedusing the 10% TMSPM monomer concentration originally selected forfurther testing based on its stability (resistance to extrusion) withlow back pressure. Once the derivatization studies had been performed,however, it was hypothesized that increasing the TMSPM monomerconcentration could further improve column capacity and extractionefficiency because of the additional surface area and silica sitesavailable for TMOS modification. Preliminary investigations had shownthat the relative column capacity of the underivatized monolith formedfrom a 17% TMSPM (peak area=54,000±5,000) was nearly double the capacityof a 10% TMSPM column (peak area=31,000±2,000). This was not trulysignificant because of the low binding capacity of the underivatizedmonoliths, but his increased number of functional sites available couldbe more important in a derivatized structure. Despite the higher backpressure associated with 17% TMSPM, the derivatization experiment wasrepeated using this monolith. The DNA profiles obtained from 10% and 17%TMSPM-modified monoliths using LIF detection are shown in FIG. 7A.Comparison of these profiles is difficult because the signal producedfrom the DNA elution peaks exceeded the detector limit. The same elutionprofiles obtained using UV detection (FIG. 7B) showed the DNA elutionpeak area produced from the 17% TMSPM column to be larger than that fromthe 10% TMSPM column, confirming the effect of monomer concentration onthe column capacity. It is noteworthy that the UV traces only providedsemi-quantitative information as the sensitivity of the UV detectionmethod was not fully assessed and the limit of detection of DNA by UV isunknown.

However, the difference in peak area observed between the 10% and 17%TMSPM monoliths is large enough to conclude that the 17% TMSPM columnhas a higher relative capacity than the 10% TMSPM monolith. In addition,the column was also found to be more robust when a higher TMSPMconcentration was used. Where pressures greater than 18 psi were foundto damage the 10% TMSPM sol-gel, the 17% TMSPM sol-gel was able towithstand pressures as high as 30 psi. This observation was importantsince the use of higher flow rates could potentially lead to shorteranalysis times.

Effect of TMSPM Monomer Hydrolysis Time on DNA Extraction Capacity. Itwas hypothesized that decreasing the pre-photopolymerization TMSPMmonomer hydrolysis time would allow for only partial hydrolysis, therebydecreasing siloxane bond formation and providing a larger number ofunhydrolyzed alkoxide groups on the monolith surface available for TMOSderivatization. However, experimental data suggested that similarrelative DNA binding capacities were achieved for full (20 min) andpartial (5 min) monomer hydrolysis (FIG. 8). One possible explanation isthat, although more alkoxide groups remain intact through the partialhydrolysis step, many of them could be buried inside the monolith afterphoto-polymerization and, therefore, inaccessible for TMOSderivatization. An alternative explanation is that even the 5 minutehydrolysis step fully hydrolyzes all of the alkoxide groups, but thedecreased time for formation of the silane bonds between monomersresults in a different sol-gel structure with an equivalent surface areaof siloxane groups. It was observed that shorter hydrolysis timeresulted in a decrease in the back pressure of the underivatizedmonolith (from 17 psi to 11 psi) for the same monomer concentration andcolumn length—this suggested that larger pores bad been formed in theshorter hydrolysis time monolith, which would allow for a higher flowrate to be applied. However, the back pressure was similar after TMOSderivatization (18 psi). It was also observed that TMOS derivatizationwas not as reproducible on columns prepared from a 5 minute monomerhydrolysis compared to columns produced with a 20 minute monomerhydrolysis (important variation in the length of the derivatizedportion). Columns produced from the long monomer hydrolysis alsodisplayed a slightly higher binding capacity as shown by the largeramount of extracted DNA, and better reproducibility (RSD=3.7% comparedto 14.4% obtained with the short hydrolysis column).

Decreasing the Monolithic Column Length. The ultimate goal of this workis to utilize these photopolymerizable sol-gels in microchips forselective placement of the DNA extraction matrix in specific regions ofan integrated processing device. To evaluate the potential of thedeveloped monolith for DNA extraction in a micro-scale format, theeffect of decreasing the monolith column length was explored. Using theoptimized monolith formulation and derivatization procedure determinedabove, the relative capacity obtained with a 12 cm monolithic column wascompared to that obtained with a 2 cm monolith (FIG. 9). Although thereduction in monomer hydrolysis time from 20 to 5 minutes did not appearto improve the column binding capacity in previous experiments, largerpores were formed (as suggested by the decrease in back pressure) andthe additional ‘space’ created within the monolith structure shouldallow for a larger number of TMOS molecules to react with each other andform a denser TMOS layer with a higher surface area available for DNAbinding. Both 5 and 20 min TMSPM hydrolysis times were, therefore,investigated again on a 2 cm column to optimize DNA binding capacity onthe shorter monolith. FIG. 9 showed that although the length of theshort monolith is 17% of the 12 cm column, the DNA recovered isapproximately 30% of the DNA eluted from the long monolith. Theseresults imply that the 12 cm column was not fully derivatized with TMOS,which is consistent with the microscopic examination of the capillaryrevealing that only part of the monolith appeared to be derivatized. Itwas also observed that decreasing the length of the column led to asignificant decrease in back pressure (from 18 psi on the 12 cm monolithto 2.5 psi on the 2 cm with 20 min TMSPM hydrolysis time and 2 psi with5 min hydrolysis time, enabling higher flow rates to be applied.Additionally, a slightly higher binding capacity was obtained from the 2cm column produced with a 5 minute monomer hydrolysis compared to a 20minute hydrolysis, suggesting that TMOS derivatization was indeed betterwhen applied onto larger pores. In order to further evaluate thepotential of the column for clinical applications, a purified humangenomic DNA sample was extracted using the optimized monolith on a 2 cmcolumn (FIG. 10). Quantification of the elution fractions by Picogreen®showed that 79.6±1.5 ng human genomic DNA had been extracted, with arelative extraction efficiency of 85.7±2.2%. These data suggest therelatively column capacity to be high, and confirm the potential of theoptimized monolith for DNA extraction in microdevices.

Evaluation of Column Efficiency Compared to Commercial SPE Methods. Inorder to assess the performance of the monolith, extractions ofpre-purified human genomic DNA and whole blood samples were performedusing the capillary-based monolith and the QIAGEN spin column. The samemass of DNA was loaded onto both columns to compare the efficiency ofthe two extraction methods. Results, displayed in Table 1, demonstratedthat the monolithic system had an extraction efficiency superior to theQIAGEN spin column for the extraction of pre-purified human genomic DNA,with extraction efficiency as high as 86% compared to 49% obtained withthe QIAGEN column. This was repeated with whole blood as the sample toassess the potential of this monolith for clinical and forensicapplications. A small volume of blood (equivalent to 83 nL of wholeblood and 4.9 ng DNA) was loaded to investigate the efficiency of thesamples where small amounts of blood (or DNA) may be involved. Theresulting data, given in Table 1, showed that DNA recovery was higherwith the monolith than with the QIAGEN spin column, mirroring withresults with pre-purified human genomic DNA. This was an importantfinding with respect to the forensic application, where analysis of afraction of a drop of blood on cloth may be required. Under theseconditions, the ability to handle low-volume, low-mass samples (a fewnanograms of DNA) is paramount. We did find, however, that the monolithwas less efficient with larger volumes of blood and did not performed upto the standards of the QIAGEN spin column which is designed for largervolumes of blood. The basis of this appears to be rooted in the surfacearea, pore size and capacity of the monolith, which appears to be lesstolerant of the tens of micrograms of protein present per microliter ofblood. However, as shown here, for the low volume samples for which thiscolumn is designed, these are not issues and the extraction efficiencyexceeds that of the commercial kit.

Although certain presently preferred embodiments of the invention havebeen specifically described herein, it will be apparent to those skilledin the art to which the invention pertains that variations andmodifications of the various embodiments shown and described herein maybe made without departing from the spirit and scope of the invention.Accordingly, it is intended that the invention be limited only to theextent required by the appended claims and the applicable rules of law.

1. A method for isolating nucleic acid on a monolithic column, saidmethod comprising the steps of: (a) preparing a monolithic column havingthe steps of (i) providing a vessel; (ii) conditioning the interiorsurface of said vessel by contacting it with a silica-based flushingsolution; (iii) preparing a silica-based monomer solution throughhydrolysis of a silica monomer; (iv) forming a polymerizable mixture byadmixing said silica-based monomer solution, an initiator material, anda porogenic solvent; (v) introducing said polymerizable mixture into thevessel; (vi) initiating the in situ polymerization of said polymerizablemixture by exposing selected portions of the mixture to UV light,thereby forming the UV photo-polymerized silica-based monolithic columnwithin portions of the vessel; (vii) flushing a silica-based monomerreagent through said UV photo-polymerized silica-based monolithiccolumn, thereby forming the grafted porous UV photo-polymerizedsilica-based monolithic column within portions of the vessel; and (b)isolating the nucleic acid on the monolithic column.
 2. The method ofclaim 1, wherein said silica-based flushing solution comprises a silicamonomer.
 3. The method of claim 2, wherein the silica monomer is3-(Trimethoxysilyl)propyl methacrylate.
 4. The method of claim 1,wherein the silica-based monomer solution comprises a silica monomer. 5.The method of claim 4, wherein the silica monomer is selected from agroup consisting of 3-(Triethoxysilyl)propyl methacrylate and3-(Trimethoxysilyl)propyl methacrylate, and3-[Tris(trimethylsiloxy)propyl methacrylate.
 6. The method of claim 5,wherein the silica monomer comprises an acrylate group joined with asilane group.
 7. The method of claim 6, wherein the silica monomercomprising an acrylate group joined with a silane group is3-(Trimethoxysilyl)propyl methacrylate.
 8. The method of claim 1,wherein the period for hydrolysis on the silica monomer ranges from 45seconds to 20 minutes.
 9. The method of claim 1, wherein thesilica-based monomer solution is present in an amount about 10 vol % toabout 50 vol %.
 10. The method of claim 1, wherein said initiation isaccomplished by a photoinitiator.
 11. The method of claim 10, whereinsaid photoinitiator is selected from the group consisting ofbenzophenone, dimethoxyacetophenone, xanthone, thioxanthone, andmixtures thereof.
 12. The method of claim 1, wherein said porogenicsolvent is an organic solvent.
 13. The method of claim 12, wherein saidporogenic solvent is an organic solvent selected from the groupconsisting of hydrocarbons, alcohols, ketones, aldehydes, organic acidesters, soluble polymer solutions, and mixtures thereof.
 14. The methodof claim 13, wherein said porogenic solvent is toluene.
 15. The methodof claim 12, wherein the solvent is present in an amount from about 70vol % to about 90 vol %.
 16. The method of claim 1, wherein theinitiating step is achieved by irradiation with ultraviolet light. 17.The method of claim 1, wherein said silica-based monomer reagentcomprises a silica monomer.
 18. The method of claim 17, wherein saidsilica monomer is selected from a group consisting oftetraethylorthosilicate and tetramethylorthosilicate.
 19. The method ofclaim 18, wherein said silica monomer is tetramethylorthosilicate. 20.The method of claim 1, wherein said silica-based monomer reagent ispresent in an amount from about 50 vol % to 90 vol %.
 21. The method ofclaim 1, wherein said silica-based monomer reagent is flushed throughthe silica-based monolith column for about 15 minutes to 120 minutes.22. The method of claim 1, wherein said vessel is a capillary, pipette,tube, or microchannel on a microfluidic device.
 23. The method of claim22, wherein said vessel is a capillary
 24. The method of claim 23,wherein said capillary is a fused silica capillary equal to 2 cm inlength, having an inside diameter of 250 μm, and an outside diameter of365 μm.
 25. The method of claim 1, wherein step (b) comprising: (i)loading a sample in the presence of a chaotropic agent onto the columnfor the binding of the nucleic acid to the column; (ii) washing thecolumn with a wash buffer solution; and (iii) releasing the boundnucleic acid from said column.
 26. The method of claim 25, wherein thenucleic acid is DNA or RNA.
 27. The method of claim 25, wherein thereleasing step is accomplished by washing the column with a buffersolution that is compatible with PCR reactions.
 28. The method of claim27, wherein the buffer solution is Tris(hydroxymethyl)aminomethane, orTris hydrochloride with EDTA.
 29. The method of claim 1, wherein thevessel is on a microchip.
 30. The method of claim 29, wherein saidmicrochip is a microfluidic device.